Abstract ADAMTS metalloproteases are prominent in arthritis as aggrecan-degrading enzymes (aggrecanases), with ADAMTS5 being a major target for drug development. ADAMTS9 and ADAMTS20 are the most highly conserved members of this protease family, and are evolutionarily related to Gon-1, a worm metalloprotease essential for gonadal morphogenesis. We identified new singular and cooperative functions of Adamts5, Adamts9 and Adamts20 in the previous funding period. Adamts9 null mice die without gastrulating and Adamts20 mutant mice (belted, bt) have defective melanoblast colonization of skin. bt/bt;Adamts9+/- mice die at birth with a complete cleft of the secondary palate, and show greater impairment of pigmentation than bt mice, indicating cooperative roles of ADAMTS9 and ADAMTS20. bt/bt; Adamts5-/- mice and Adamts9+/-;Adamts5-/- mice develop soft-tissue syndactly (STS), owing to failure of apoptosis during interdigital web regression. STS in these combinatorial mutants is present at a higher frequency than in Adamts5, Adamts9, or bt mice alone. All three proteases process the proteoglycan versican. Versican cleavage is reduced in bt skin and bt/bt;Adamts5- /- webs during their regression, and Vcan interacts genetically with these proteases in STS and cleft palate. Moreover, biochemical studies and genetics identified fibulin-1 as a co-factor for ADAMTS5 in web regression. Rescue of STS by exogenous BMP4 suggests impairment of BMP signaling in the absence of the ADAMTS proteases. Hypothesis: ADAMTS5, ADAMTS9 and ADAMTS20 cooperate within specific molecular networks, which contain fibulins and versican, to influence signaling in craniofacial and limb development. Specific aims: 1: To determine the role of Adamts9 in craniofacial and limb development. 2. To define molecular mechanisms of syndactyly in Adamts deficient mice. 3: To uncover the operational mechanisms of ADAMTS proteases by characterizing versican processing and fibulin interactions. Significance: Understanding the fundamental biology of limb and craniofacial development is key to several common congenital malformations, such as cleft palate. ADAMTS5 is a major therapeutic target in arthritis;understanding its normal function and mechanisms, as well as of closely related proteases, is thus essential for insights on potential side effects and off-target effects of inhibitors, and for development of new therapeutic approaches.